This invention pertains to a novel liquid egg yolk product which contains lysophospholipoprotein. More particularly, this invention pertains to a novel liquid egg yolk product containing lysophospholipoprotein from a phospholipoprotein modified using a non-animal derived phospholipase A, and a process therefor, which is kosher, does not have a porcine or bovine source, and does not contain appreciable levels of amylase. The product is useful as an emulsifier in foodstuffs such as sauces, spreads, mayonnaise, dressings, salad dressings, and the like.
Phospholipases are enzymes which act on phospholipids which are found in animal and vegetable cells. Phospholipases are selective enzymes which are classified according to their site of action in the phospholipid molecule. Thus, a phospholipase A1 hydrolyzes the bond between the fatty acid and the glycerine residue at the 1-position of the phospholipid.
The hydrolysis of a phospholipid by a phospholipase results in the production of a xe2x80x9clysophospholipidxe2x80x9d. Although phospholipids have many industrial uses, lysophospholipids have been shown to be particularly suitable for certain industrial applications. Lysophospholipids have a high solubility in water and this property gives them enhanced emulsification properties in oil/water emulsions. Lysophospholipids have an ability to form emulsions which are reasonably stable to changing pH conditions, including acid conditions, and they are resistant to changing temperatures. The ability of the lysophospholipid to form an oil-water or water-oil emulsion is not reduced by the presence of ions, such as magnesium or calcium ions.
The foregoing properties of the lysophospholipids make them particularly desirable for use in the food, cosmetics and pharmaceutical industries. It has been demonstrated that the conversion of a phospholipid to a lysophospholipid in a phospholipid containing substance, such as a food product, generally leads to an improvement in the stability of that substance.
The most commonly used phospholipase in the industrial hydrolysis of phospholipids is pancreatin, which is an enzyme prepared from the pancreas of pigs. Enzymatic hydrolysis of a phospholipid, using a phospholipase isolated from a micro-organism is, however, known. Such hydrolysis using a phospholipase A is described, for example, in Japanese Unexamined Patent Publication No. Sho-58-212783, and the hydrolysis using a lipase is described in Japanese Unexamined Patent Publication No. Sho63-42691. Furthermore, the enzyme Taka-Diastaseh(trademark), which was isolated from a species of Aspergillus, A. oryzae, [Biochem. Z., 261 (1933) 275], has demonstrated a lipase activity which is capable of hydrolyzing a phospholipid. The enzymes isolated from microorganisms have been shown to have less activity than porcine pancreatic enzyme. Moreover, the microorganisms produce amylase and protease as by-products, which are undesirable because they break down starch and proteins and lead to emulsion instability.
Although pancreatin has better properties than enzymes isolated from microorganisms, hydrolysis of a phospholipid using pancreatin has many disadvantages. Firstly, it may be necessary to make continual adjustments to the pH of the reaction mixture during hydrolysis of a phospholipid substrate with porcine pancreatin. The optimum pH for activity of pancreatin is in the range from neutral to weakly alkaline. During the hydrolysis reaction, however, the release of free fatty acids causes the pH to drop, that is, it increases the acidity of the reaction mixture, so that unless counter action is taken, the mixture will become acidic, and therefore outside the pH range for optimum activity of the enzyme.
Traditionally, heat treatment has been used to deactivate the residual enzyme in processes involving the use of enzymes. However, porcine pancreatin has another disadvantage because it is not fully deactivated by heat treatment, and even treatment of the enzyme at a temperature of 95xc2x0 C. for 30 minutes may not sufficiently deactivate the residual enzyme. The use of a higher temperature is impossible in view of the sensitivity of the phospholipid and free fatty acids to heat.
FEMS Microbiol. Lett. 3(2), 85-7, Vol. 3, No. 2, 1978 discloses the detection of phospholipase A1 activity in various filamentous fungi, including Aspergillus strains, but there is no disclosure of the isolation and purification of the enzyme. Biological Abstracts, vol. 72, Philadelphia, Pa., Abstract No. 012592, discloses the purification and characterization of phospholipids by various phospholipases.
It is known from British patent specification GB-B-1,525,929 (Unilever) to treat phospholipoproteins or phospholipoprotein containing materials, such as egg yolk, whole egg, blood serum, wheat protein, soybean, and the like, with phospholipase A. The phospholipase A is also active when the phospholipid is complexed with protein. After the treatment with the phospholipase, the lysophospholipoprotein is formed. The lysophospholipid is complexed with a protein. The lysophospholipoprotein containing material disclosed in GB-B-1,525,929 has achieved considerable commercial success as an emulsion stabilizer, particularly in oil-in-water emulsions. They enabled the manufacture of sterilizable emulsions, which in practice turned out to be commercially very successful, because they had a long shelf life and an excellent creamy taste.
The following patents disclose subject matter which is related to or relevant to the subject invention.
Japanese Abstract No. 58212783 A2, Kyowa Hakko Kogyo Co. Ltd., discloses a process whereby a microorganism, e.g. Streptomyces scabies ATCC15485 or Streptomyces achromogenes variety streptozoticus NRRL2697, belonging to the genus Streptomyces, and having the ability to produce phospholipase A, is cultivated in a culture medium at 22xc2x0 C. to 40xc2x0 C. and a neutral or slightly alkaline pH for about 2 to 6 days. The phospholipase A is collected mainly from the culture fluid.
Japanese Abstract No. 06153939 A2, Snow Brand Milk Prod. Co. Ltd., discloses a process whereby an alga of the genus Euglena (preferably Euglena gracilis) having the ability to produce phospholipase A is cultured in a culture medium containing a carbon source (preferably glucose), a nitrogen source (preferably glutamic acid or diammonium hydrogenphosphate) at 4-35 ratio (C/N) under conditions of preferably pH 3.0-4.5, 20-32xc2x0 C. culture temperature and irradiation with light or in the dark for 3-7 days, to produce and accumulate phospholipase A in the organism. The resultant phospholipase A is then separated and collected to provide the objective phospholipase A.
U.S. Pat. No. 5,521,080, Hattori et al., discloses a method for preparing a phospholipase A1 which comprises (a) culturing a phospholipase A1 producing strain of Aspergillus under conditions which allow for the production of the phospholipase A1; (b) after culturing, diluting the culture with water or an appropriate buffer solution; (c) filtering the resulting solution under pressure to remove any insoluble matter; and optionally (d) purifying the enzyme.
U.S. Pat Nos. 5,378,623 and 5,538,874, Hattori et al., are related and disclose a phospholipase A1 which is capable of hydrolyzing a phospholipid to produce a 2-acyl lysophospholipid and is obtainable from species of the fungus Aspergillus.
EP 0 575 133 B1, Sankyo Company Limited, discloses a phospholipase A1 obtainable from fungus selected from Aspergillus niger and Aspergillus oryzae characterized in that said phospholipase A1: (a) hydrolyzes phospholipid between about pH 2.5 and about pH 6.0; (b) has a molecular weight of between about 30,000 and about 40,000 daltons, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis; (c) has a stability to temperature with an upper limit of between about 45 and about 90xc2x0 C.; (d) has a pI under isoelectric point electrophoresis at about pH 2.8 to about pH 4.5; and (e) has an optimum temperature for activity of from about 30 to about 65xc2x0 C.
U.S. Pat. No. 4,119,564, van Dam, discloses a process whereby oil-in-water emulsions with an increased viscosity are produced by incorporating an effective amount of phospholipase A-treated phospholipoprotein.
Related U.S. Pat. No. 4,034,124, van Dam, discloses emulsions comprising an oil phase, an aqueous phase and a phospholipoprotein which has been subjected to a treatment with phospholipase A as an emulsion stabilizer. These emulsions have an increased stability, especially heat stability, compared with emulsions which do not contain such a stabilizer.
U.S. Pat. No. 5,028,447, Schenk, discloses a process whereby oil and water emulsions which contain a phospholipoprotein material which has been modified by phospholipase A, and at least one native starch based thickening agent, are prepared by subsequently gelatinizing the thickening agent, incorporating the modified phospholipoprotein containing material into the gelatinized thickening agent, then incorporating the oil (which may at least partially be replaced by a low-calorie fat substitute) and finally homogenizing the mixture obtained. Canadian Patent No. 1,210,224 is related.
EP 0 319 064 B1, Unilever Nev., discloses a process for the preparation of a water and oil emulsion comprising a phospholipoprotein containing material, which has been modified by phospholipase A, and at least one native starch based thickening agent, which comprises: (a) at least partly gelatinizing the native starch based thickening agent; (b) incorporating the phospholipoprotein containing material, which has been modified by phospholipase A, into the gelatinized native starch based thickening agent; (c) incorporating from 5% to 85% by weight of oil or fat containing oil into the mixture obtained in step (b); and (d) homogenizing the final mixture obtained.
U.K. Patent No. 1,585,105, Unilever Limited, discloses an oil-in-water emulsion which contains a phospholipase A-treated phospholipoprotein having a degree of conversion of at least 55% and at least one thickening agent in a proportion which is less than that required for obtaining an emulsion of the same composition and viscosity but containing phospholipoprotein of a lower degree of conversion.
U.S. Pat. No. 5,082,674 and Canadian Patent No. 2,026,447, Carrell et al., disclose a process for the manufacture of a lysophospholipoprotein-comprising foodstuff. The dried lysophospholipoprotein or dried lysophospholipoprotein-comprising material, preferably having a moisture content of at most 10 wt %., at a level of 0.1-90 wt. % therein, is used as a texture-modifying agent, a glossing agent, a freeze-thaw stabilizing agent, a heat-stabilizing agent and a syneresis-inhibiting agent.
U.S. Pat. No. 5,314,706, Colarow et al., discloses an egg yolk fortified with exogenous soybean lysophosphatidylcholine contained in exogenous soybean lysophospholipids which is employed as an emulsification agent in oil and water emulsions, particularly in emulsions which are sterilized. The agent may be obtained by hydrolyzing phospholipids derived from soybeans with phospholipase A2, deactivating the phospholipase A2 with a proteolytic enzyme and then inactivating the proteolytic enzyme by heat-treatment at a temperature of from 80xc2x0 C. to 90xc2x0 C. Egg yolk is fortified by combining and homogenizing the so-obtained lysophospholipids, or exogenous phospholipids containing lysophosphatidylcholine.
U.S. Pat. No. 5,750,164, Saito et al., discloses a method of decreasing cholesterol concentration in eggs, processed egg foodstuffs, meat, fish meat, dairy products and processed foodstuffs thereof, which includes hydrolyzing phospholipids in the eggs or processed egg foodstuffs or other products with one member selected from the group of phospholipase A1, A2, B, D, lysophospholipase and a mixture thereof, and subjecting the phospholipid-hydrolyzed eggs or processed egg foodstuffs or other products to a conventional cholesterol-decreasing treatment.
Japanese patent Abstract No. 62262998 A2, QP Corp., discloses a process whereby a natural phospholipid-containing substance such as egg yolk is added with a phospholipase A2 preparation-containing material) (e.g. purified phospholipase A2 preparation originated from animal pancreas) to effect enzymatic reaction. The phospholipid in the substance is decomposed by the reaction to obtain a lysophospholipid containing material. The obtained lysophospholipid-containing material is dried at about s 80xc2x0 C. by spray drying, etc., to powder having a water content of s 10%. The powder is extracted with a polar solvent such as ethanol to extract lysophospholipid. The solvent is distilled from the extract under reduced pressure to obtain the objective lysophospholipid-containing material composed of 68 wt. % neutral lipid and 32 wt. % phospholipid (30 wt. % thereof is lysophospholipid).
Japanese patent Abstract No. 63209742 A2, QP Corp., discloses a method whereby an emulsifier is prepared by mixing phospholipid, protein, phospholipase A2 and clean water uniformly in a mixer or a colloid mill, etc., treating at ca. 40xc2x0 C. and drying if necessary. Suitable protein is albumin, globulin, gelatin, etc., and suitable proportion of protein to be present in aqueous suspension of phospholipid is 1:(ca 0.5 to 2) phospholipid to protein. The phospholipase A2 is an enzyme for hydrolyzing the fatty acid ester moiety at the middle part of a glyceride constituting the phospholipid, and the amount thereof to be used is ca. 0.1 to 5 wt. % based on the amount of the phospholipid.
U.S. Pat. No. 5,213,968, Castle et al., discloses a process whereby emulsifying agents are prepared by sequentially treating a biological material with a protease and a lipase. The enzymatically treated biological material may be pasteurized during or following the enzymatic treatment.
EP 0 414 024 B1, Societe des Produits Nestle S.A., discloses a process for the preparation of an emulsifying agent which comprises treating a biological material containing a lipid as well as a lipoprotein and/or a protein with a protease and a lipase and pasteurizing the product. The treatment with the protease and the lipase is carried out sequentially in any order.
The present invention relates to a process for the manufacture of lysophospholipoprotein-comprising foodstuffs, such as sauces, spreads, mayonnaise, dressings, soups, bakery products, creamers, creamer-thickeners, ice cream, drinks, dairy products, desserts, sherbets, meals, and combinations thereof, with no detectable amylase (and protease) activity.
The invention is directed to a process for the manufacture of a liquid egg yolk product containing lysophospholipoprotein comprising: (a) processing a phospholipase A-containing microbial fermentate to remove undesirable-amylase and protease co-products of the fermentation to produce a refined phospholipase A-containing microbial product; and (b) combining a liquid egg yolk with the refined phospholipase A-containing microbial product of step (a) to produce a modified liquid egg yolk product containing lysophospholipoprotein, said modified liquid egg yolk product having (i) a degree of conversion of phospholipoprotein to lysophospholipoprotein of at least 10%; and (ii) an amylase activity of less than 50 units/litre.
The phospholipase A-containing fermentate of step (a) can be produced by a fermentation of a phospholipase A-producing microorganism in a nutrient medium. Suitable prior art phospholipase A producing microorganisms can be used in the invention but a preferred microorganism can be Streptomryces violaceoruber or a genetically modified Thermontyces lanuginous/Fusarium oxysporun. Step (a) can be conducted at a temperature below about 20xc2x0 C. and at a pH between about 4.9 and 5.2.
The undesirable amylase and protease co-products can be removed in step (a) by passing the fermentate through a cross-flow dialysis apparatus having a PES 50K membrane.
The liquid egg yolk product containing lysophosphoprotein can have a phospholipase activity of less than 250 units/litre. The liquid egg yolk product containing lysophospholipoprotein can have a protease activity of less than 0.01 fluorescence units/mi/min.
The invention is also directed to a product prepared according to the process of the invention. The product can contain up to 20% added salts, and up to 50% added carbohydrates, including sugar, malto-dextrin, glucose or corn syrup solids. The product can contain up to 75% added liquid egg albumin or concentrated liquid egg albumin.
The product can be spray dried at an inlet temperature of from about 200xc2x0 C. to about 250xc2x0 C. and an air outlet temperature from about 75xc2x0 C. to about 100xc2x0 C. so that the final moisture of the dried product is less than about 10% by weight.
The product can be incorporated into an emulsion of oil, water, vinegar, starch, sugar and salt. The emulsion can include egg yolk.